Proteomics Analysis of Rat Brain Postsynaptic Density

Abstract

The postsynaptic density contains multiple protein complexes that together relay the presynaptic neurotransmitter input to the activation of the postsynaptic neuron. In the present study we took two independent proteome approaches for the characterization of the protein complement of the postsynaptic density, namely 1) two-dimensional gel electrophoresis separation of proteins in conjunction with mass spectrometry to identify the tryptic peptides of the protein spots and 2) isolation of the trypsin-digested sample that was labeled with isotope-coded affinity tag, followed by liquid chromatography-tandem mass spectrometry for the partial separation and identification of the peptides, respectively. Functional grouping of the identified proteins indicates that the postsynaptic density is a structurally and functionally complex organelle that may be involved in a broad range of synaptic activities. These proteins include the receptors and ion channels for glutamate neurotransmission, proteins for maintenance and modulation of synaptic architecture, sorting and trafficking of membrane proteins, generation of anaerobic energy, scaffolding and signaling, local protein synthesis, and correct protein folding and breakdown of synaptic proteins. Together, these results imply that the postsynaptic density may have the ability to function (semi-) autonomously and may direct various cellular functions in order to integrate synaptic physiology.The majority of excitatory neurotransmission in the brain occurs via glutamatergic synapses. In the presynaptic element of the synapse, specialized secretion machinery determines the activity-dependent membrane fusion of glutamate-containing vesicles and the release of transmitter into the synaptic cleft. In the postsynaptic element, glutamate receptors and downstream signal transduction are organized by the protein assembly of the postsynaptic density (PSD). 1 Both the presynaptic release machinery and the PSD are electron-dense assemblies (1, 2), in which proteins are thought to be organized into distinct functional complexes (3-5) that may be dynamically regulated by neuronal activity (6 -8). The modulation of this molecular architecture of the synapse is at the basis of synaptic plasticity (6 -8), and aberrations thereof may underlie neuronal disorders.In view of the importance of the PSD in glutamatergic neurotransmission and its involvement in neuroplasticity, considerable efforts have been made to identify its protein constituents as a prelude to understand the molecular basis of PSD functioning. In the past several years, yeast two-hybrid technology has been extensively used to characterize proteins that interact with the glutamate receptors and may constitute core elements of the PSD involved in the regulation of receptor trafficking and signaling (reviewed in Refs. 9 -11). Based largely on these studies a protein-protein interaction map of the PSD has emerged. In brief, the model posits that the postsynaptic receptor complexes are localized by scaffolding proteins such as the s...

Mass of the postsynaptic density and enumeration of three key molecules

“…This estimate is almost identical to the one based on measurements of NMDA channel conductance (49,50) and open probability P 0 Ϸ 0.3 (51): Nϫ P 0 Ϸ 8, N Ϸ 27 (52). If a PSD were composed solely of molecules of Ϸ100 kDa and included Ϸ100 copies of each type of protein, then there should be Ϸ100 types of proteins in the PSD, which is in line with current estimates (11)(12)(13)(14).…”

“…A PSD fraction isolated from forebrain contains many different macromolecules, including receptors, scaffolding molecules, kinases, phosphatases, and cytoskeletal components (9), in particular, N-methyl-D-aspartate receptors (NMDARs), ␣-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid receptors (AMPARs), and scaffolding molecules, such as PSD-95, synapse-associated protein (SAP)97, Shank, and Homer (10). Proteomic analysis by mass spectrometry of the PSD fraction puts the possible varieties of PSD proteins at Ϸ100-400 (11)(12)(13)(14). This large number of associated protein species implies a complexity in the structure of the PSD, where positioning and numbers of copies of individual molecules are functionally important (15).…”

Insulin receptor substrate of 53 kDa links postsynaptic shank to PSD‐95

“…Via its PDZ domains, PSD-95 is one of the major anchoring proteins for postsynaptic transmitter receptors and ion channels (Kim et al 1995;Kornau et al 1995). Through an intricate network of protein interactions, a large protein complex of up to 100 proteins is assembled at the spine heads around the PSD-95/transmitter receptor complex (Husi et al 2000;Walikonis et al 2000;Li et al 2004) which has been termed the postsynaptic density (PSD). The function of the PSD appears to be to physically link postsynaptic receptors to signalling molecules, and to provide stable attachment of the receptors to the actin-based cytoskeleton of the dendritic spine.…”

Different protein profiles in inferior colliculus and cerebellum: A comparative proteomic study

“…Reduction of the sample complexity by pre-fractionation represents a possible solution to this problem. Several pre-fractionation protocols have been developed that are applicable to brain tissue, including those suitable for plasma membrane proteins, which may be most interesting to neuroscientists (Li et al, 2004;Guillemin et al, 2005;Schindler et al, 2006Schindler et al, , 2008. Separating the proteome into less complex sub-fractions is generally limited by the amount of starting tissue available, as each separating step involves loss of proteins.…”